PROJECT SUMMARY/ABSTRACT Patients with acute respiratory distress syndrome (ARDS) often require mechanical ventilation (MV), which further induces lung injury (ventilator-induced lung injury, VILI). VILI is associated with substantial morbidity and mortality both in mechanically ventilated patients with, and without ARDS. Pneumonia (PA) is a primary risk factor for development of ARDS in humans. In rodents, VILI and PA have been extensively used as experimental acute lung injury (ALI) models to study the pathogenesis of ARDS. The scientific premise of this grant is that receptor-interacting protein-3 kinase (RIPK3) is crucial for the propagation of necroptotic cell death and inflammation in the pathogenesis of experimental ALI (VILI and PA models) and in human disease such as ARDS. We have obtained intriguing preliminary data demonstarting that RIPK3 expression is regulated in ALI, and that RIPK3-deficient mice are protected against ALI (in both VILI and PA models), strongly suggesting that the RIPK3-dependent necroptosis pathway is crucial for mediating the pathogenesis of experimental ALI. We further show that metabolic dysfunction such as dysregulated fatty acid (FA) metabolism results in activation of RIPK3-dependent signaling and necroptosis and that disruption of FA metabolism promotes macrophage inflammasome activation and pro-inflammatory cytokines production, which contributes to the development of ALI. We also show also that FA synthesis and mitochondrial NADPH:oxidase-4 (NOX4) are required for NLRP3-mediated inflammasome activation in macrophages. In human studies, we demonstrate that not only are inflammasome regulated cytokines associated with mortality of the critically ill patients but both necroptosis proteins RIPK3 and FA are regulated in patients with critical illness. Based on these studies, we propose the following hypothesis: Sterile injurious mechanical ventilation or pneumonia infection causes metabolic and FA dysfunction resulting in activation of RIPK3-dependent signaling and necroptosis. Disruption of FA metabolism by mechanical injury or infection promotes macrophage NOX4-dependent inflammasome activation and pro-inflammatory cytokines production, which contribute to the development of ALI. We also hypothesize that necroptosis-related proteins and FA are associated with morbidity and mortality in patients with critical illness, including ARDS. We will test our hypothesis by addressing the following Specific Aims: Specific Aim 1: To determine the regulation and function of RIPK3-dependent necroptosis in ALI. Specific Aim 2: To determine the mechanisms by which necroptosis mediates NOX4-dependent NLRP3 inflammasome activation in ALI. Specific Aim 3: To determine the clinical relevance of necroptosis and FA metabolism in the critically ill patients, including ARDS.